Spirulina Phycobiliproteins as Food Components and Complements

Spirulina has a documented history of use as a food for more than 1000 years, and has been in production as a dietary supplement for 40 years. Among many of Spirulina bioactive components, blue protein C-phycocyanin and its linear tetrapyrrole chromophore phycocyanobilin occupy a special place due to broad possibilities for application in various areas of food technology. The subject of this chapter is up-to-date food applications of these Spirulina components, with a focus on their use as food colorants, additives, nutriceuticals, and dietary supplements. Their other actual and future food application possibilities will also be briefly presented and discussed

Probing the stability of the food colourant R-phycoerythrin from dried Nori flakes

The high content of vitamins, minerals, antioxidants, and proteins makes red algae Porphyra sp. (Nori) superfood with exceptional health-promoting benefits. Its intense colour originates from R-phycoerythrin (R-PE), phycobiliprotein containing covalently attached tetrapyrrole chromophores: red phycoerythrobilin and orange phycourobilin. The present study aims to characterize the stability of R-PE, a natural colourant with a high potential for application in the food, cosmetic, and pharmaceutical industries. We purified R-PE from dried Nori flakes with a high purity ratio (A560 /A280 ≥5). Far-UV CD spectroscopic showed that α-helix is the dominant secondary structure (75%). The thermal unfolding of α-helix revealed two transitions (Tm1 and Tm2 at 56 and 72°C, respectively), ascribed to the different subunits of R-PE. Absorption measurements showed that high pressure (HP) induces dissociation of R-PE into subunits followed by subunit unfolding. Contrary to temperature, HP treatment showed a significant advantage under applied conditions: the protein unfolding is partly reversible, and the R-PE colour bleaching is minimized. Based on the fluorescence quenching approach, R-PE's binding affinities for Cu2+ and Zn2+ ions were 6.27x105 and 1.71x103 M-1, respectively. Absorption and near-UV/VIS CD spectroscopy suggested conformational changes in protein chromophores upon metal ions binding. Far-UV CD spectroscopy did not reveal that metal binding affects R-PE structure. The obtained results give new insights into the stability of R-PE with a good use-value in replacement of toxic synthetic dyes, preservation of R-PE red colour in fortified food and beverages by HP processing, and as a biosensor for Cu2+ in aquatic life systems. Acknowledgments: This study was financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Contract number: 451-03-9/2021-14/200168 and the European Commission, under the Horizon2020, FoodEnTwin Project, GA No. 810752

The effects of biliverdin on pressure-induced unfolding of apomyoglobin: The specific role of Zn2+ ions

Apomyoglobin (apoMb), a model protein in biochemistry, exhibits a strong propensity to bind various ligands, which makes it a good candidate as a carrier of bioactive hydrophobic drugs. The stability of its hydrophobic pocket determines its potential as a carrier of bioactive compounds. High pressure (HP) is a potent tool for studying protein stability, revealing the specific role of hydrophobic cavities in unfolding. We probed the effects of biliverdin (BV) binding and its complex with Zn 2+ ions on the structure and HP stability of apoMb. CD spectroscopy and SAXS measurements revealed that BV and BV-Zn 2+ complexes make the apoMb structure more compact with higher α-helical content. We performed in-situ HP measurements of apoMb intrinsic fluorescence to demonstrate the ability of BV to stabilise apoMb structure at HP conditions. Furthermore, the presence of Zn 2+ within the apoMb-BV complex significantly enhances the BV stabilisation effect. In-situ visible absorption study of BV chromophore confirmed the ability of Zn 2+ to increase the stability of apoMb-BV complex under HP: the onset of complex dissociation is shifted by ~100 MPa in the presence of Zn 2+. By combining HPfluorescence and HP-visible absorption spectroscopy, our strategy highlights the crucial role of tetrapyrrole-metal complexes in stabilising apoMb hydrophobic pocket

Digestion by pepsin releases biologically active chromopeptides from C-phycocyanin, a blue-colored biliprotein of microalga Spirulina

C-phycocyanin, the major protein of cyanobacteria Spirulina, possesses significant antioxidant, anti-cancer, anti-inflammatory and immunomodulatory effects, ascribed to covalently attached linear tetrapyrrole chromophore phycocyanobilin. There are no literature data about structure and biological activities of released peptides with bound chromophore in C-phycocyanin digest. This study aims to identify chromopeptides obtained after pepsin digestion of C-phycocyanin and to examine their bioactivities. C-phycocyanin is rapidly digested by pepsin in simulated gastric fluid. The structure of released chromopeptides was analyzed by high resolution tandem mass spectrometry and peptides varying in size from 2 to 13 amino acid residues were identified in both subunits of C-phycocyanin. Following separation by HPLC, chromopeptides were analyzed for potential bioactivities. It was shown that all five chromopeptide fractions have significant antioxidant and metal-chelating activities and show cytotoxic effect on human cervical adenocarcinoma and epithelial colonic cancer cell lines. In addition, chromopeptides protect human erythrocytes from free radical-induced hemolysis in antioxidative capacity dependant manner. There was a positive correlation between antioxidative potency and other biological activities of chromopeptides. Digestion by pepsin releases biologically active chromopeptides from C-phycocyanin whose activity is mostly related to the antioxidative potency provided by chromophore. (C) 2016 Elsevier B.V. All rights reserved.Peer-reviewed manuscript: [http://cherry.chem.bg.ac.rs/handle/123456789/3444]Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3445

Exploring and strengthening the potential of R-phycocyanin from Nori flakes as a food colourant

This study aimed to purify, characterise and stabilise the natural food colourant, R-phycocyanin (R-PC), from the red algae Porphyra spp. (Nori). We purified R-PC from dried Nori flakes with a high purity ratio (A618/A280 ≥ 3.4) in native form (α-helix content 53%). SAXS measurements revealed that R-PC is trimeric ((αβ)3) in solution. The thermal denaturation of α-helix revealed one transition (Tm at 52 ◦C), while the pH stability study showed R-PC is stable in the pH range 4–8. The thermal treatment of R-PC at 60 °C has detrimental and irreversible effects on RPC colour and antioxidant capacity (22 % of residual capacity). However, immobilisation of R-PC within calcium alginate beads completely preserves R-PC colour and mainly retains its antioxidant ability (78 % of residual capacity). Results give new insights into the stability of R-PC and preservation of its purple colour and bioactivity by encapsulation in calcium alginate beads

Noncovalent interactions of bovine α-lactalbumin with green tea polyphenol, epigalocatechin-3-gallate

Bovine alpha-lactalbumin (ALA) is an important Ca-binding protein of milk. Epigallocatechin-3-gallate (EGCG) is the major and the most biologically active catechin of green tea, which has the highest binding affinity to whey proteins due to galloyl functional group. In this study experimental and computational methods were used to investigate noncovalent interactions of EGCG and ALA. Binding affinity of EGCG for ALA, determined by fluorescence quenching analysis, was in the range described for complexes of EGCG and other dietary proteins, and lower than affinity of some phenolic compounds to ALA. Based on circular dichroism and Fourier transform infrared spectroscopy spectra, binding of EGCG change ALA conformation inducing alpha-helix to beta-structures transition. The isothermal titration calorimetry results suggest that the binding of EGCG to ALA is enthalpically favorable. The docking analysis shows that EGCG binds in the hydrophobic pocket at the entrance of cleft between alpha-helical and beta-sheetrich domains and includes residues of aromatic cluster II. Uptake of ALA by monocytes proceeds at a slower rate in the presence of EGCG suggesting that EGCG binding may impair uptake of ALA by antigen-presenting cells. ALA, being of low cost and widely available protein, can serve as suitable delivery system for EGCG, as well as for food fortification with this bioactive catechin. (C) 2016 Elsevier Ltd. All rights reserved.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3585

(A) Temperature dependence of 0.5 μM HSA ellipticity at 222 nm in the presence and absence of 0.5 μM PCB; (B) Temperature dependence of 1 μM HSA fluorescence at 340 nm in the presence and absence of 1 μM PCB (λ_EX = 280 nm); (C) SDS-PAGE profile after trypsin digestion of 3.8 μM HSA in the presence and absence of 3.8 μM PCB.

<p>Lane K: HSA without trypsin; lane M: MW markers; lanes 1–6 correspond to digestion times 0.5, 2, 5, 10, 30, and 60 min without PCB, respectively; lanes 7–12 correspond to digestion times 0.5, 2, 5, 10, 30 and 60 min with PCB, respectively.</p

A) PCB chemical structure with labeled (A-D) pyrolle rings (oxygen atoms are in red and nitrogen in blue); B) Schematic of HSA structure with domains and ligand binding sites labeled; Radius of gyration (Rg) values (C) and 2D RMDS plots for free HSA (D), HSA-PCB(IIA) (E), and HSA-PCB(IB) (F) during 300 ns molecular dynamic simulation.

<p>A) PCB chemical structure with labeled (A-D) pyrolle rings (oxygen atoms are in red and nitrogen in blue); B) Schematic of HSA structure with domains and ligand binding sites labeled; Radius of gyration (Rg) values (C) and 2D RMDS plots for free HSA (D), HSA-PCB(IIA) (E), and HSA-PCB(IB) (F) during 300 ns molecular dynamic simulation.</p

Covalent binding of food-derived blue pigment phycocyanobilin to bovine β-lactoglobulin under physiological conditions

In this study, we investigated structural aspects of covalent binding of food derived blue pigment phycocyanobilin (PCB) to bovine beta-lactoglobulin (BLG), major whey protein, by spectroscopic, electrophoretic, mass spectrometry and computational methods. At physiological pH (7.2), we found that covalent pigment binding via free cysteine residue is slow (k(a)=0.065 min(-1)), of moderate affinity (K-a=4x10(4) M-1), and stereo-selective. Binding also occurs at a broad pH range and under simulated gastrointestinal conditions. Adduct formation rises with pH, and in concentrated urea (k(a)=0.101 min(-1)). The BLG-PCB adduct has slightly altered secondary and tertiary protein structure, and bound PCB has higher fluorescence and more stretched conformation than free chromophore. Combination of steered molecular dynamic for disulfide exchange, non-covalent and covalent docking, favours Cys119 residue in protein calyx as target for covalent BLG-PCB adduct formation. Our results suggest that this adduct can serve as delivery system of bioactive PCB

Catalogue Maison Bigot [titres “Viens dans ma nacelle” => “Bazeilles”]

Catalogue Maison Bigot, 157 rue du Temple [Paris] : “Romances et chansons nouvelles” (verso “Sommes nous prêts ?”) ; titres : “Viens dans ma nacelle => “Bazeilles” ; quatre colonnes par deux foix (titres, auteurs, compositeurs, genres) ; interprètes crédités dans la colonne “genre” : Marius Richard, Mercadier, Amiati, Debailleul, Paulus, B. Delahaye, Gibert, Garnier [Léon Garnier], d’Aubreuil [Daubreuil ?], Antony [Antony Lassaigne], Duhem [Émile Duhem], Canon [Fernande Canon], Bourgès, Bonnet, E. Faure [Elise Faure], Dlle (Mlle?) Derly, Libert, Caudieux, Leduc, Judith, G. Lange [Gabrielle Lange], Mme Barnoll, Kaiser [Augustine Kaïser], Limat [Louis Limat], Farville, Réval [Jules Réval], Velly [Louis Velly], Plessis [Henri Plessis] ; datation par analyse des titres 1886 (hypothèse à confirmer)